KR20100026270A - Method and apparatus for multiplex detection based on dielectrophoresis and magnetophoresis - Google Patents

Abstract

PURPOSE: A method and a device for multiplex detection using dielectrophoresis and magnetophoresis are provided to improve separation efficiency by using dielectrophoretic force. CONSTITUTION: A method and a device for multiplex detection using dielectrophoresis and magnetophoresis comprises an electrode array(110), a fluid channel(100), and a magnetic field inclination generator(120). The electrode array comprises more than two electrodes. The fluid channel is arranged crossing the electrode array. The magnetic field inclination generator is arranged on or under the fluid channel to be not overlapped with the electrode array.

Description

METHOD AND APPARATUS FOR MULTIPLEX DETECTION BASED ON DIELECTROPHORESIS AND MAGNETOPHORESIS}

The present invention relates to methods and apparatus for multiple detection and separation of species, including biomass. Specifically, the present invention relates to a technique for multiple detection and separation of micro-scale magnetic particles or biomaterials bound thereto from a single sample solution using the techniques of genophoresis (DEP) and magnetophoresis (MAP).

Separation of cell types or intracellular components is required as a preparative tool for diagnosis and treatment in the medical field, and for final purposes or other assays in the field of research. There are many types of cell sorting methods currently used in laboratories and clinical laboratories. The rapid isolation of other kinds of particles, such as viruses, bacteria, cells and multicellular entities, is a central step in various applications in the fields of pharmaceutical research, clinical diagnostics and environmental analysis. Rapidly growing knowledge in drug discovery and protein research is enabling researchers to gain a greater understanding of protein-protein interactions, cell signaling pathways, and markers of metabolic processes. This information is difficult or impossible to obtain using single protein detection methods, eg, traditional methods such as ELISA or Western blotting.

Because microfluidics reduces the time and cost associated with conventional analyses while improving reproducibility, recent efforts have been made to convert conventional biological operations into lap-on-a-chip systems. Characterization of biomass and most important tools to improve the efficiency of the preparation steps are the ability to recognize and selectively manipulate, interact and / or sequester the target component in the mixture. In addition, microbead-based assays have advantages over flat microarrays. Efficient cleaning, multiple analyzes using encrypted microbeads, short signal analysis time due to large signal to volume amplification and free movement of microbeads in the media.

Separation performance is assessed by three characteristics: "Throughput" refers to how many analytes can be identified and sorted per unit time, and "purity" refers to the fraction of the target analyte in the trapping region. "Recovery rate" means the fraction of the injected target analyte that has been successfully classified into the trapping region. Fluorescence activated cell sorter (FACS), dielectrophoretically activated cell sorter (DACS) and magnetically activated cell sorter (MACS) have been used for cell separation and manipulation. . These techniques provide high specificity for cell separation, but have drawbacks. For example, FACS has a limited throughput (mostly 103-104 cells / second). In addition, the sorting time is long, the mechanical stress of the nozzle is significant, so the cell viability is reduced, and the cellular functional survival rate is also reduced. It is also expensive and complex in design and operation.

WO 01/96857 A2 relates to a method comprising the positioning of particles to be separated, which is controlled within a hydrodynamic flow profile using opposite electrophoretic and magnetic force balances. In this case, the process of changing the field over time during separation, called "programming", is essential to improve the separation efficiency. Magnetic field programming is also essential to achieve flexibility and differentiation. In addition, the sample analyte mixture must spend sufficient time in the DEP and MAP fields to allow the analytes to move closer to position in the carrier fluid flow profile and thereby be separated from each other. Problems involved in the manufacture of such DEP-MAP-FFF devices are the electrode and magnetic pole arrangements.

Summary of the Invention The present invention seeks to provide a method and apparatus for multiplex detection and separation of species, which achieves high throughput, purity and separation efficiency by using the electrophoretic and magnetophoretic forces in the fluid channel. It is also an object of the present invention to provide a method and apparatus for separating biological materials useful for multiple detection in microfluidic devices or lab-on-chip.

The present invention includes an electrode array (110) comprising two or more electrodes; A fluid channel 100 disposed in a direction crossing the electrode array; And a magnetic field gradient generator 120 disposed so as not to overlap with the electrode array above or below the fluid channel.

The present invention also provides a method for detecting multiple species using the device, comprising: injecting a sample comprising two or more species into a fluid channel; Injecting the liquid carrier media into the fluid channel; Applying an alternating voltage to the electrode array; Boosting the material species in the sample to different heights in a channel with a dielectrophoretic force generated from the electrode array; And moving the supported species to a magnetic field region in the channel and trapping at one position with a magnetophoretic force.

By using the separation method and the device according to an embodiment of the present invention, a large amount can be separated in parallel at low cost, the size of the device is small, it is disposable and can be used clinically practical. The separation method and apparatus can be applied promisingly in the fields of biology, medicine and materials engineering to therapeutic uses such as detecting multiple markers for the early detection of diseases and identifying rare cells, as well as characterizing micro / nanoparticles.

Multiple detection, separation method and apparatus according to an embodiment of the present invention using the electrophoretic and magnetophoretic forces in the fluid channel, by applying a different electrophoretic force depending on the particle type to support the particles to different heights, the magnetic field It enters the area where the change occurs and causes the particle to trap at a specific location within the channel with a magnetophoretic force. In addition, the multiple detection, separation method and apparatus according to an embodiment of the present invention, if the particles of the same size is not separated by the electrophoretic force, by arranging the particles of the same size to support the same height by acting the electrophoretic force After this, the magnetic field may be accurately separated according to the magnetic characteristics in the region where the magnetic field change occurs.

In the multiple detection, separation method and apparatus according to one embodiment of the invention, the Dielectrophoretic force (F _DEP ) can be adjusted by the applied alternating frequency. In its simplest form, a electrophoretic separator simply separates specific particles from the media flowing across an array of electrodes. The particles are separated at a rate based on frequency and flow rate from carrier media on the electrode array. In this way, particles that experience negative electrophoresis (DEP) are separated from particles that experience positive DEP. Depending on the particle type, different DEP forces can be exhibited when the same frequency is applied. When a component experiences positive DEP, they move in a direction very close to the electrode array. When experiencing negative DEP, they move away from the electrode array. This flotation height difference allows components to enter the magnetophoretic region at different heights.

로 예상될 수 있다. (1)μ_o는 자유 공간의 투과성이고, B는 자속밀도, Δχ = χ_particle-χ_medium 는 미디어에 대한 입자의 자화율 차이(differential magnetic susceptibility), 그리고 V_m는 상자성 입자의 부피이다. 따라서, 힘은 자속밀도 B의 제곱의 기울기에 기초한다. 많은 응용에서, 초상자성 입자가 포화 상태에 있을 때, 총 자화부피

는 일정하고 초상자성 입자에 대한 자기영동 힘에 대한 방정식은

로 단순 화될 수 있다. 여기에서 m_sat 는 상기 입자의 포화 자화정도이다. 초상자성 입자에 대한 힘의 방향 및 크기가 인가된 자기장의 경사에 의해 조절되기 때문에, 자기영동 분리 장치는 이러한 파라미터를 정확하게 조절할 수 있도록 설계될 수 있다. In the separation method and apparatus according to an embodiment of the present invention, the magnetic field gradient generated by magnetic field gradient generators (MFG) is applied to the magnetophoretic force, which acts on the magnetic particles in the microfluidic device. F _MAP ). In weak diamagnetic media, such as most buffer solutions, the magnetophoretic forces for paramagnetic particles

Can be expected. (1) μ _o is the permeability of free space, B is the magnetic flux density, Δχ = χ _particle -χ _medium is the differential magnetic susceptibility to the media, and V _m is the volume of paramagnetic particles. Therefore, the force is based on the slope of the square of the magnetic flux density B. In many applications, the total magnetization volume when the superparamagnetic particles are in saturation

Is a constant, the equation for the magnetophoretic forces for superparamagnetic particles is

Can be simplified. Where m _sat is the degree of saturation magnetization of the particles. Since the direction and magnitude of the force on the superparamagnetic particles are controlled by the inclination of the applied magnetic field, the magnetophoretic separation device can be designed to precisely control these parameters.

The direction and magnitude of the magnetic field gradient generated through the MFG is based on the structure of the MFG and the applied magnetic field (typically provided by an external magnet in proximity to the isolation region). Suitable parameters of the MFG structure include the MFG material, the size and arrangement of the MFG, the flow of the MFG and the direction of the external magnetic field.

The material from which the MFG composition is made must have a permeability significantly different from that of the fluid media (eg buffer) in the device. MFG compositions can be made from ferromagnetic materials. Thus, the MFG composition may comprise at least one of iron, cobalt, nickel samarium, disporium, gadolinium, or an alloy of other components that together form a ferromagnetic material. The material may be a pure material (eg nickel or cobalt) or may be a ferromagnetic alloy such as an alloy of copper, manganese and / or tin. Examples of suitable ferromagnetic alloys include Heusler alloys (65% copper, 25% manganese and 10% aluminum), Permalloy (55% iron and 45% nickel), Supermalloy (15.7% iron, 79% nickel, 5 % Molybdenum and 0.3% manganese) and μ-metal (77% nickel, 16% iron, 5% copper and 2% chromium). Nickel-cobalt alloys may also be used. In some embodiments, ferrite, which is a mixture of nonmetal ferromagnetic materials, iron and other metal oxides, may also be used.

In multiple detection, separation methods and apparatus according to one embodiment of the present invention, labels and analytes will generally impart dielectric and magnetic properties to the label-analyte complex based on their relative permittivity, permeability, and volume. . Since the separation described herein is based on flotation and trapping by electrophoretic and magnetophoretic forces, the label-analyte complexes in DEP-MAP separators have different effluent and other effluent characteristics of labels that contain other analytes. Indicates.

The method according to one embodiment of the invention allows the labels to be separated and separated according to their DEP forces (positive / negative). For example, cells bound to different numbers of magnetic labels will exhibit different DEP forces during separation, thus allowing for differentiation, identification, and separation. As another example, labels that are not associated with an analyte can be separated from labels associated with cells, particle molecules and other target analytes.

In addition, "cocktails" consisting of different types of labels can be made, each label type having a different genetic and magnetic "fingerprint". Each label type in the mixture can be distinguished, separated, identified and characterized independently depending on the binding status with the different target analytes. Such label cocktails enable simultaneous analysis of multiple analytes in the mixture in a single separation step.

In the analysis sequence before the step where the group of particles is trapped at a specific position in the magnetic region of the microchannel, the dielectric properties of the particles enable automatic separation of the solid phase from the liquid phase at the applied frequency. The separation can be used for a variety of purposes. Removal of sample debris from analyte, removal of sample components that are a significant cause of background noise in the detection phase, removal of competing binding elements that are not analyte and may interfere with results, and labeling, analysis Such as removing bound species from unbound species, such as water, analyte binding elements, and label-binding element binders.

The particular function in a particular assay or combination of assays will depend on the assay characteristics and assay protocol. Manipulating magnetic or magnetizable objects means moving magnetic or magnetizable objects, mixing different forms of magnetic or magnetizable objects, separating different types of magnetic or magnetizable objects from each other, magnetic or It means to move the magnetizable object away from the surface of the device or to the surface. Apparatus and methods in accordance with embodiments of the present invention can be used in combination with the manipulation of a magnetic or magnetizable object in combination with detecting the presence or determining the concentration of a magnetic or magnetizable object in a sample fluid. While the prior art provides this separation and detection in a slow, complex approach, the present invention allows for the separation and detection of multiple labels at a faster rate from a single sample solution. Fast analysis is especially necessary for the detection of many viruses.

DEP is a very efficient way to manipulate and isolate species. This technique mainly applies to cells, organelles and other particles (eg cell contents and membranes). When a particle is placed in an electric field, charge is induced due to the relative permittivity and conductivity of the particle compared to the media. This process is called polarization. If the external electric field is not uniform, the particles can move by the electrostatic force. In particular, in an alternating electric field the particle polarization is frequency dependent so that the induced force and thus the particle movement can be adjusted. This is called genetic electrophoresis (DEP). By varying the induced force, the particles can either approach or repel by the conventional DEP, or they can be moved in both directions by the traveling wave DEP. DEP can also be used to identify or separate other particles. (E.g., other types of bacteria, living cells or dead cells), the main advantage of DEP is that the mobility and hence the mode of transport can be controlled by a single electric field.

However, DEP performance is very sensitive to fluids such as buffers, in particular ionic strength. Large DEP forces can only be obtained in low ionic strength media. On the other hand, the ionic strength of real samples such as blood is much higher. Moreover, since DEP force strength is usually proportional to the volume of the particles, it is only suitable for the manipulation of large particles such as cells and is too small to manipulate small molecules. In addition, DEP of a bioanalyte does not necessarily reflect the biological properties of the analyte, but has a large physical effect. Thus, it can be difficult to manipulate analytes with specific specificities in complex environments.

Another method for bioanalyte migration is the use of magnetophoresis (MAP). Magnetic particles can be operated by magnetophoretic force. The advantage is that biospecificity is maintained by biocompatible binding between the magnetic particles and the bioanalyte. Another advantage is that the magnetophoretic force is not affected by the media. Since most media do not contain magnetic components, it is easy to integrate magnetic sensors with microsystems with detection, which is useful for lab-on-chip (LOC) applications.

In the uneven electric field generated by the interdigitated electrode arrangement, the neutral particles migrate because of their polarization effect. This phenomenon is genetic electrophoresis. One embodiment of the present invention provides a detection method based on the fact that the labels can be genetically arranged or sorted and then the path can be altered within the microchannels when the magnetic field induces magnetization on the particles.

From a simple single detection method, multiple detection techniques are needed because it is very difficult to understand the mechanisms for protein-protein interactions, cellular signal transduction pathways. The present invention relates to a multiple detection system based on DEP-MAP.

One embodiment of the invention discloses a method of distinguishing molecules or cells in one sample. The method is a method in which hydrodynamic forces are adjusted to DEP forces on a molecule or cell, thereby selectively moving the molecule or cell in a desired direction in the fluid flow. In one embodiment, the desired direction includes moving the molecule or cell from the first region of the fluid flow to the next fluid flow region. In another embodiment, the molecule or cell comprises a naturally occurring DEP fingerprint. In another embodiment, the molecule or cell can be modified with a DEP tag. DEP forces may be generated by one or more electrodes. The method can be performed in a microfluidic device. The method allows the sorting of multiple labels from heterogeneous mixtures. This method is reliable and has good applicability.

Exemplary electrode arrangements for electrophoretic manipulations can be in any form, such as engagement form and sex form. In one embodiment, the electrode array may be a flat array consisting of a thin film of conductor, such as platinum, gold, chromium, etc., patterned on a supporting substrate such as glass. This arrangement can be manufactured by known photolithography techniques and electron beam (e beam) pattern techniques. Such an electrode arrangement, or other arrangement, may be used to provide DEP forces in the classifier according to one embodiment of the present invention. The electrodes can be electroplated, printed or etched or attached to the chamber wall. Or, when located in or around the flow channel and connected to the signal generator, may generate an uneven electric field in the flow channel. In one embodiment, an insulating layer may or may not be used, depending on the solution used as the carrier media. The insulating layer is not limited to SiO ₂ , Si ₃ N ₄ .

In conventional immunoassays and separations with magnetic labels, magnetic fields were used to capture all labels and fluorescent signals were used to measure the amount of antigen present in the sample. However, in one embodiment of the present invention a magnetic field was used to detect biomolecules in the microfluidic channel. In addition, supersensitive magnetic biosensors using superconducting quantum interference devices (SQUIDs) have been developed for immunoassay, but the disadvantage of this method is a single analysis based on the distinction between bound and unbound magnetic particles. It is limited to water analysis only. In one embodiment of the present invention, a small volume of samples can be mixed with a heterogeneous label mixture to capture a particular analyte from one sample. In one embodiment of the present invention, it is possible to distinguish magnetic particles of various sizes encoded with different and more specific analytes.

In order to form a strong magnetic field gradient in the trapping region, the magnetic field may be provided by a permanent magnet or a permanent magnet adapted to MFG. In one embodiment, the external magnet may be a permanent magnet or an electromagnet (eg Helmholtz coil. In general, the electromagnet produces a smaller magnetic field but can be designed to form a very even field. May be advantageous.

The position and direction in the trapping region of the permanent magnet may be determined by the strength of the magnetic field formed by the permanent magnet, the uniformity of the field (ie, the uniformity of the field across the trapping region in the absence of MFG), the dimension and shape of the magnet, and the like. In one embodiment, the magnet may be located above and below the microfluidic channel. One embodiment uses a single magnet with a single pole located above or below the trapping region. Another embodiment uses multiple magnets located above or below the trapping area and having the same distance to the same pole.

As described above, the method according to one embodiment of the present invention not only separates magnetic particles having different characteristics from each other at a slow flow rate, but also strong and delicate separation that can be achieved at high flow rates to increase throughput. System.

We have designed a microfluidic device for separating labels of various sizes. The microfluidic device includes three functions and each unit: a concentrated flow region, a dielectric domain (DEP) region, and a magnetic domain (MAP) region. In the concentrated flow zone, the labels are hydrodynamically concentrated in a controlled width flow, while in the DEP zone, an unequal electric field is generated by the electrode array to facilitate separation and the separated labels are separated by permanent magnets located in the MAP zone. Trapped. The DEP region and the MAP region are located at two different positions in the same microchannel. Based on the size of the label, the DEP forces acting on the label may vary. This separates the labels. If the same size labels are not separated by the DEP force, the DEP force may be applied to float the labels of the same size to the same height, and then may be accurately separated according to the magnetic characteristics in the MAP region.

Positive DEP occurs when the particles are more polarizable than the media so that the particles are attracted toward the higher inclined region. Negative DEP occurs when the particles are less polarizable than the media and are attracted towards areas of lower inclination.

When the magnet is located under the microchannel so that the electrode array is on the same side as the magnet, the device is said to be under "negative DEP" mode. This magnet position promotes labels that have experienced negative DEP to move towards the opposite end of the magnet. Labels experiencing positive DEP here are aligned towards the other half of the microchannel. Similarly, when the magnet is on the opposite side of the electrode array, labels that have experienced positive DEP immediately gather on the magnet. Labels experiencing negative DEP, on the other hand, move further away without gathering at near magnet ends. The positions of these magnet and electrode arrays form a "positive DEP" mode. The possibility of these two different modes is possible as an embodiment of the separation method of the present invention.

Although the term "microfluid" is used in the above description, the principles and design features described herein may be applied to larger scale devices and systems using millimeter or centimeter scale channels. Although one channel or station is described herein, the channel or location may be an example of multiple channels or locations arranged to operate in parallel on a single substrate or multiple substrates of an integrated system. The microfluidic "system" may include one or more microfluidic devices and associated fluidic connections, electrical connections, controls, and the like. Microfluidic devices include the presence of passages, eg channels, through which one or more fluids in the microfluidic dimension flow. Magnetic labeling technology is one of the methods performed to achieve the above-mentioned process. The "magnetic label" or "label" described above is a magnetically susceptible solid phase particle, which may be microbead. Because the magnetic microbeads have a large surface area per unit volume, they provide a relatively large binding site to which a target particle, cell or molecule (“target analyte”) can bind. Thus, magnetically sensitive labels with modified surfaces tend to stick to specific target analytes in the analyte mixture. "Electrode" means all electrical passages, eg conductor arrangements.

1 shows three components on an integrated chip in an apparatus according to one embodiment of the invention. Component 100 represents a fluid channel and component 110 represents an electrode arrangement providing a DEP force. Component 120 represents a magnetic field gradient generator that enables label collection, and component 130 represents a fully integrated microfluidic device. Magnetic particles are represented by component 140.

The microfluidic device 130 includes a substrate (substrate 180 may be one or more substrates connected to each other to define a fluid channel boundary therebetween). The sample inlet 150 is in fluid communication with the carrier media inlet 170. Although FIG. 1 shows three fluid inlets, the device according to one embodiment may include a single fluid inlet and a flow channel. The outlet is indicated by component 160.

In one embodiment, substrate 180 is an insulating (eg glass or polymer), or semiconducting (eg, glass or polymer) in which various forms of microfluidic device 130 (eg, channels, chambers, valves, etc.) are designed. For example silicone based).

DEP is the transfer of charge neutral material caused by the polarization effect in an uneven electric field. Due to the relatively easy operation of the electric field and the interface to integrated electronics, DEP is particularly advantageous for on-chip label manipulation. DEP forces are due to an uneven distribution of alternating electric fields on which species (eg, prokaryotic, eukaryotic, viral, molecular, particle, and multicellular individuals) lie. In particular, DEP forces are caused by the interaction between the polarized charge induced by the electric field and the unequal electric field. Polarization charges are induced in the magnetic particles 140 by the applied field, and the strength and direction of the generated dipoles are related to the difference in the genophoretic properties between the material species and the floating media.

The DEP force may be a traveling-wave dielectrophoresis (twDEP) force or a conventional dielectrophoresis (cDEP) force. The twDEP force refers to the force on the magnetic particles 140 generated from the traveling wave electric field. The traveling wave electric field is characterized by an alternating electric field component having an uneven distribution in phase values. The cDEP force refers to the force on the magnetic particle 140 generated from an uneven distribution in the strength of an alternating electric field.

By changing the phase of the alternating electrical signal, a second DEP force, namely traveling wave DEP, is generated. The cDEP force depends on the spatial inequality of the electric field and causes the particles to move in the direction away from or near the region of high field strength. The twDEP force depends on the phase distribution of the applied electric field and causes the particles to move in a direction away from or in the direction of increasing phase value.

Dielectrophoretic fields are generated by applying a voltage between two or more electrodes. Typically the electrodes are arranged in such a position that the applied electric field may be uneven or cause spatial variation to produce a dielectrophoretic effect. Thus, the particles can be manipulated by changing the electrophoretic field. The same frequency can provide a system in which some components experience negative DEP and others experience positive DEP.

In order to efficiently separate the desired species from the complex mixture, in one embodiment, the electrophoretic amplitude response of the target analyte can be controlled by labeling the analyte with magnetic particles 140 of different sizes to maintain different polarization reactions. .

The fact that both the magnitude and polarity of the DEP force at the applied frequency varies with each particle type has significant significance in using alternating DEP for selective manipulation of bioparticles and separation of bioparticles. This is because different particles may have different DEP reactions at the same electric field frequency.

From the equation, it can be seen that the DEP force is proportional to the particle volume, so that the particles can be separated according to their size. In alternating electrophoresis the force can be controlled by the frequency of application. The force is cube dependent on magnitude, linear dependence on permittivity, and magnitude and polarity of force are dependent on frequency. One embodiment of the present invention uses this feature to design a flow through system for label separation.

The method and apparatus according to one embodiment of the present invention is designed and constructed to take advantage of the differences in genophoretic response between labels with different genophoretic fingerprints, or between unlabeled cells and particles and labeled cells or particles. It became.

In addition, the magnetic field gradient generator 120 may include applying an external magnetic field from a permanent magnet or an electromagnet.

Permanent magnets are produced from nickel, cobalt, iron, alloys thereof, and alloys of nonferromagnetic materials that become ferromagnetic by alloying, ie, alloys known as Heusler alloys (eg, alloys of copper, tin, and manganese). Many suitable alloys for permanent magnets are known and commercially available for making magnets that can be used in one embodiment of the present invention. Typical such materials are transition metal-semimetal (metalloid) alloys, with about 80% transition metals (usually Fe, Co, or Ni) and semimetal components that lower the melting point (boron, carbon, silicon, phosphorus or aluminum). Is made from Permanent magnets can be crystalline or amorphous. One example of an amorphous alloy is Fe ₈₀ B ₂₀ (Metglas 2605).

In the embodiment of FIG. 1, the external magnetic field is caused by rectangular (10 cm × 2.5 cm × 1 cm) NdFeB magnets (K & J Magnetics, Jamison, PA) attached to the top and bottom surfaces of the fractionation region of the microfluidic device 130. Is provided.

Magnetic capture particles used in the microfluidic separation of the present invention may take many different forms. In one embodiment it may be superparamagnetic microparticles, nanoparticles, in another embodiment it may be ferromagnetic or paramagnetic.

As a general suggestion, the magnetic particles 140 should be selected to have a size, mass and magnetization that can be easily redirected from the direction of fluid flow when exposed to the magnetic field in the microfluidic device 130 (hydraulic and magnetic Balance of effects). In one embodiment, the magnetic particles 140 do not retain magnetism when the magnetic field is removed. In a typical example, magnetic particles 140 include iron oxides (Fe ₂ O ₃ and / or Fe ₃ O ₄ ) having a diameter of about 10 nanometers to about 100 nanometers. However, in other embodiments, larger magnetic particles 140 may also be used. For example, it is possible to use magnetic particles 140 large enough to be used as support media for cells in culture.

Magnetic particles 140 may be coated with a material that can be compatible with the microfluidic environment and coupled with a particular target analyte. Examples of coatings include polymer shells, glass, ceramics, gels, and the like. In one embodiment, the coating is coated with materials that promote coupling or physical bonding with the target analyte. For example, the polymer coating on the micro magnetic particles may be coated with an antibody, nucleic acid sequence, avidin or biotin.

The first group of magnetic particles 140 may be nanoparticles such as those available from Miltenyi Biotec Corporation of Bergisch GladBach, Germany. These are relatively small particles made from coated single domain iron oxide particles, typically in the range of about 10-100 nanometers in diameter. These are coupled to specific antibodies, nucleic acids, proteins and the like.

The second group of magnetic particles 140 is made from magnetic nanoparticles embedded in a polymer matrix such as polystyrene. These are typically smooth and generally spherical. Suitable beads are available from Invitrogen Corporation, Carlsbad, CA. These magnetic particles 140 are also coupled to specific antibodies, nucleic acids, proteins, and the like.

In one embodiment, the intrinsic magnetism of the sample material may be used. In this case, the magnetic particles 140 need not be used. Examples of such materials include erythrocytes, industrial small magnetic particles, and the like.

Commonly used sorting buffers include phosphate buffered saline, deionized water and the like. The actual buffer composition will depend on the application and the nature of the sample and target analyte. In one embodiment used to classify bacteria, the buffer comprises 1 × PBS (phosphate buffered saline) / 20% glycerol / 1% BSA (bovine serum albumin) (volume basis).

The fluid channel 100, sample inlet 150, carrier media inlet 170, vias, pumps, etc. required for the microfluidic detection location of the present invention may be fabricated using known fabrication techniques (e.g., For example, it can be manufactured according to various microfabrication procedures). In one embodiment, a borosilicate glass wafer may be secured to a PDMS replica of a silicon master mold fabricated by applying a precursor to the silicon master and curing it. Plasma bonding may be used to bond the glass substrate 180 to the PDMS layer.

The fluid flow can be driven by pressure or by electrodynamics. The pressure-driven flow is generated by pumps (eg peristaltic pumps), syringes, etc., which are readily available for application to small volumes of microfluidics. In one embodiment, a programmable syringe pump (Harvard Apparatus PHD. 22/2000, Holliston. MA) is used to deliver the sample mixture and fractionation buffer at a constant flow rate into the microfluidic device 130. The flow of the sample in the microchannels can be monitored through a suitable detector such as an inverted microscope (eg Olympus, Japan) with a CCD camera.

The electrode array 110 is a planar array composed of thin film conductors such as platinum, gold, and chrome patterned on a supporting substrate 180 such as glass. This arrangement can be fabricated using known photolithography and e-beam pattern techniques. Such an electrode array 110, or other type of electrode array, may be used to provide DEP forces in the separator according to one embodiment of the present invention. The electrodes can be electroplated, printed, etched or fixed to the channel wall, or located in or around the flow channel to produce an uneven electric field in the flow channel when coupled to the signal generator. In one embodiment, an insulating layer may or may not be used depending on the solution used as the carrier media. The selection of the insulating layer is not limited to SiO ₂ , Si ₃ N _{4, or the} like.

Assume that an uneven electric field is generated by applying an alternating voltage to the electrode array 110. 1 shows an electrode array 110 comprising a conductor (eg, a thin film of gold, chromium, etc.) patterned on a non-conductive substrate (eg glass). If an electrical voltage is applied between the electrodes when the electrode arrays 110 are parallel, an electric field in which the peripheral effect is spatially uneven above and below the plane of the electrode is created. The electrophoretic force experienced by particles located in this surrounding electric field region will vary with the distance from the electrode array 110 plane to the particles.

In one embodiment of the invention, of the microparticles, magnetic microparticles may be used. In one embodiment, the invention can combine multiple heterogeneous binding analysis of a single fluid sample by flow cytometry with the use of solid magnetic particles as the solid phase to facilitate separation of the solid and liquid phases. Such microparticles provide various choices for the differential parameters described above and are easily analyzed and distinguished by flow cytometry. Magnetic microparticles also offer the possibility of separation from the liquid phase by a magnetic field. The groups do not overlap substantially, and the average value of the characteristic characteristics of the adjacent groups is sufficiently separated from the other groups so that the groups can be distinguished by conventional automated detection methods. In one embodiment, the analytical reactant may be bound to each particle, substantially all of the particles in each group include the same analytical reactant, and each group includes a different analyte reactant than the adjacent group. Thus, the groups can be distinguished not only by their characteristic differential parameters but also by analytical reactants bound to the particles. Magnetic separation is also useful in the washing step and for recovering the supernatant from the first solid phase after the captured antibody has been separated. The present invention can provide a new analysis format for building multiple analysis platforms in a lab-on-chip.

Hereinafter, a preferred embodiment of the present invention regarding the separation of particles and biomass will be described in more detail. However, the following embodiments are merely preferred embodiments of the present invention, and the present invention is not limited to such embodiments.

A schematic diagram of an apparatus according to an embodiment of the present invention is shown in FIG. A cross-sectional view of the device in operation is shown in FIG. 2A. When the magnetic particles 140 flow across the electrode, F _DEPz acts along the z direction, and the applied flow rate acts along the y direction of the magnetic particles 140. After the magnetic particles experience the dielectrophoretic force F _DEPz exerted by the electrode array 110, the height in their fluid channel 100 is varied. Then they enter the magnetic migration area along the z direction at different heights and there, but the F _DEPz contrast to experience the magnetic migration force F _MAPz. Although the applied speed is V, component 140 has different speed factors as V ₁ and V ₂ in the DEP area and the MAP area, respectively. Here, the magnetic particles 140 are collected at a specific position in the fluid channel 100 by the magnetic field generated by the magnetic field gradient generator 120.

2B shows that as soon as the flow rate is applied by the programmable syringe pump, the magnetic particles 140 of different sizes achieve concentrated flow along the fluid channel 100.

2C shows the separation of magnetic particles 140 of different sizes based on DEP forces in the DEP field region. In the absence of the magnetic field gradient generator 120, the magnetic particles are not trapped in the MAP region. Instead, the particles flow along the channel because the flow rate acting on the magnetic particles 140 is dominant.

2D shows that after magnetic particles 140 experience DEP forces, they enter the MAP region at different heights. The magnetic field generated by the magnetic field gradient generator 120 causes the magnetic particles 140 to trap at different specific locations within the fluid channel 100.

1 is a diagram showing each component of an apparatus according to an embodiment of the present invention.

2A is a cross-sectional view of a device in operation in accordance with one embodiment of the present invention.

FIG. 2B is a diagram showing that particles achieve a concentrated flow along the channel as soon as a flow rate is applied.

2C is a plot showing the separation of particles based on DEP forces in the DEP field region.

2D is a plot showing that particles enter the MAP region at different heights after experiencing DEP forces.

* Description of the main parts *

100: fluid channel

110: electrode array

120: magnetic field gradient generator

130: microfluidic device

140: magnetic particles

150: sample inlet

160: outlet

170: carrier media inlet

180: substrate

Claims (18)

An electrode array including two or more electrodes; A fluid channel 100 disposed in a direction crossing the electrode array; And A device for detecting multiplex devices comprising a magnetic field gradient generator (120) disposed above or below the fluid channel so as not to overlap the electrode array. The method of claim 1, The magnetic field gradient generator (120) is disposed on the same side as the electrode array (110), or multiple substance detection device, characterized in that disposed on the opposite side of the electrode array (110). The method of claim 1, And the electrode array is an interdigitated or castellated array. The method of claim 1, And the electrode array is in the form of a thin film composed of platinum, gold or chromium patterned on a supporting substrate. The method of claim 1, The magnetic field gradient generator is composed of a ferromagnetic material, characterized in that the multi-material species detection device, characterized in that the permanent magnet or electromagnet. A method for multiplexing species species using the device of any one of claims 1 to 5, Injecting a sample comprising two or more species into a fluid channel; Injecting the liquid carrier media into the fluid channel; Applying an alternating voltage to the electrode array; Supporting material species in the sample in a channel with a dielectrophoretic force generated from the electrode array; And Moving the supported species to a magnetic field region within the channel and trapping at one location with a magnetophoretic force. The method of claim 6, Suspending the material species in the sample in the channel with the dielectrophoretic force, characterized in that to support the material species to each different height. The method of claim 6, Suspending the material species in the sample in the channel with the dielectrophoretic force, the material species multiple detection method characterized in that to arrange the material of the same size to the same height. The method of claim 6, Injecting the sample and liquid carrier media into a fluid channel, and then applying a flow velocity. The method of claim 9, The flow rate application is a multiple species detection method, characterized in that using the pump or syringe. The method of claim 6, The material species is a biomaterial (biomaterial), the sample is a multiplex detection method, characterized in that the biological sample. The method of claim 11, The biomaterial is a virus, bacteria, cells, intracellular organs, molecules or multicellular entities, characterized in that the multiple species detection method. The method of claim 6, The mass species is in a form coupled to a magnetic solid phase, wherein the solid phase is coupled to an analyte reactant which is selectively active only in the desired substance species. The method of claim 13, And said solid phase is superparamagnetic, ferromagnetic or paramagnetic. The method of claim 13, Wherein said solid phase is a particle having a diameter of 10 nanometers to 100 nanometers. The method of claim 13, The method of detecting multiple species, characterized in that the assay reagent is an antibody, nucleic acid or protein. The method of claim 13, The material species bound to the solid phase is a material species multiple detection method, characterized in that the form of a single material species bound to a plurality of solid phases. The method of claim 13, The multiple species detection method, characterized in that the solid phase to which the species are bound is one or more kinds.

Images